PROJECT SUMMARY Advanced carcinomas are among the most lethal human diseases, being metastatic prostate cancer (PC) a classic example. Despite current treatments, metastatic PC progresses to a therapy resistant stage that precedes lethality. Therefore, there is a need to identify new targetable mechanisms driving cancer aggressiveness to continue improving patients? outcome. The main objective of this research proposal is to dissect the mechanisms by which Nuclear Pore Complexes (NPCs) regulate genome functions (gene expression and integrity) in PC and uncover novel NPC-regulated targetable pathways using innovative pre-clinical models of lethal PC. NPCs contribute to different biological functions through their building blocks nucleoporins (Nups), including nucleo-cytoplasmic transport, chromatin organization, gene expression, genome integrity and mitotic regulation. In the context of cancer, Nups have been associated to tumor formation and development, however their specific mechanistic role in cancer pathogenesis remains largely unknown. Recently we identified clinically relevant Nups upregulated in lethal PC through interrogation of publicly available patient tissue sample transcriptomic datasets containing primary and castration-chemotherapy-resistant metastatic prostate tumors. Within the upregulated Nups, POM121 was shown to play a role in the aggressiveness of lethal PC (cell survival, proliferation and tumorigenesis) via nuclear transport regulation. Yet, the specific transport-dependent and - independent mechanisms by which POM121 regulates the aggressive features of PC and its crosstalk with other Nups remain unknown. Notably, functional genomic studies (transcriptomic and computational analysis) combined with biochemical and single-cell high-resolution imaging have revealed a crosstalk between two top upregulated Nups in lethal PC, POM121 and TPR, which impacts on the aggressiveness properties of PC cells. Specifically, we found that PC cell survival is enhanced by the POM121 transcriptional regulation of specific genome stability genes and TPR, which in turn regulates mitotic checkpoint activity, through Androgen Receptor (AR)-dependent and independent mechanisms. Crucially, our studies have also uncovered a potential novel role of soluble chromatin-bound Nups in the regulation of aggressive features of PC cells potentially through direct transcriptional regulation. Thus, collectively these results led to the hypothesis that NPCs regulate PC aggressiveness through multifaceted mechanisms controlling both genome functions and stability. We will address this hypothesis and determine the Nup-specific mechanisms contributing to PC aggressiveness through three aims. In Aim 1, we will define POM121 nuclear transport-dependent and -independent mechanisms promoting PC aggressiveness. In Aim 2, we will examine the POM121-TPR mechanistic interplay enhancing genome stability and cell survival in lethal PC. In Aim 3, we will evaluate the clinical significance and efficacy of targeting specific NPC-regulated pathways controlling genome integrity in patient derived pre-clinical models.